The morphology and structure of the MOFs were investigated by scanning SEM and FTIR, respectively. The performance of the activated electrode toward the electrooxidation of glucose in alkaline solution was evaluated with cyclic voltammetry technology in the potential range from 0.2 V to 0.6 V. The electrochemical activated Ni-MOFs exhibited obvious anodic (0.46 V) and cathodic peaks (0.37 V) in the 0.1 M NaOH solution due to the Ni(II)/Ni(III) transfer. A linear relationship between the glucose concentrations (ranging from 0 to 10 mM) and anodic peak currents with R2 = 0.954 was obtained. It was found that the diffusion of glucose was the limiting step in the electrochemical reaction. The sensor exhibited good selectivity toward glucose in the presence of 10-folds uric acid and ascorbic acid. Moreover, this sensor showed good long-term stability for continuous glucose monitoring. The good selectivity, stability, and rapid response of this sensor suggests that it could have potential applications in long-term non-enzymatic blood glucose monitoring.Continuous intensive monitoring of glucose is one of the most important approaches in recovering the quality of life of diabetic patients. One challenge for electrochemical enzymatic glucose sensors is their short lifespan for continuous glucose monitoring. Therefore, it is of great significance to develop non-enzymatic glucose sensors as an alternative approach for long-term glucose monitoring. This study presented a highly sensitive and selective electrochemical non-enzymatic glucose sensor using the electrochemically activated conductive Ni Diabetes, caused by insulin deficiency or resistance, is a chronic disorder of glucose metabolism. It has become one of the most widespread diseases in the world and has profound implications for disability and mortality 2, NiO, Ni(OH)2, NiS2, and ZnO, have exhibited non-enzymatic glucose sensing performance 2  MOFs exhibit very high electrical conductivity, exceeding those of previous semiconducting metal-organic graphene analogs and other conductive MOFs, which are even higher than some of the best organic conductors , which are constituted by organic linkers and metal ions or clusters, have rapidly been developed 2 MOFs was utilized as sensing materials to develop electrochemical non-enzymatic glucose sensors. The morphology and structure of MOFs were characterized by scanning SEM and FTIR, respectively. In fact, after the activation of Ni-MOFs in KOH solution, the conductive MOFs exhibited stable reduction and oxidation peaks in alkaline solution, which was very sensitive to glucose concentrations. Moreover, the activated Ni-MOFs showed excellent selectivity and stability. All the obtained results suggest the promising prospects and potential applications of this sensor for non-enzymatic and long-term blood glucose monitoring.In this study, the electrochemically activated conductive Ni2·6H2O  and Nafion (5% solution) was brought from Sigma-Aldrich. Potassium hexacyanoferrate (K4Fe(CN)6), triethylamine, glucose, KCl, KOH, uric acid (UA), and ascorbic acid (AA) of an analytical reagent were purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals were of an analytical reagent grade and were used without further purification. All solutions were prepared by distilled water and stored at room temperature.HITP·6HCl  were brought from Shanghai TenSus Biotechnology Co., Ltd. NiCl2·6H2O in 5 mL of water and 0.1 mL of triethylamine solution was added to a solution of 10 mg of HITP·6HCl in 5 mL of water. The mixture was stirred under an oil bath at 65°C for 3 h. The resulting black powder was centrifuged, filtered, and washed with water several times and then dried under a vacuum oven for 24 h.The preparation of Ni-MOFs followed previous reports with some modifications . A standard three-electrode cell was applied in all the measurements. Ni-MOFs modified glass carbon electrode (GCE) and electrochemical treated Ni-MOFs electrode were used as working electrodes. The platinum rod and saturated calomel electrode (SCE) were used as counter electrodes and reference electrodes, respectively. All the electrochemical tests were performed at room temperature.anodic-Pcathodic in 0.1 M K4Fe(CN)6/0.01M KCl below 80 mV vs. SCE. A mixture solution of 16 μL Ni-MOFs (1 mg mL−1) and 4 μL Nafion was added to the polished GCE surface and dried under room temperature. To active the Ni-MOFs, the cyclic voltammogram method was performed at the potential between 0.2 V to 0.6 V vs. SCE at a scan rate of 50 mV s−1 for 100 cycles in a 0.1 M KOH solution until stable curves were obtained.The GCE was polished with 1.5 μm, 0.5 μm, and 50 nm aluminum oxide sequentially until the P−1. The selectivity testes were applied in the mixture glucose with 10-fold concentration of AA and UA.The activated Ni-MOFs electrode was investigated as a glucose sensor in an alkaline medium. Cyclic voltammograms (CVs) measurements were processed in 0.1 M KOH in the potential range from 0.2 to 0.6 V with a scan rate of 50 mV sThe morphology was observed by a scanning electron microscope . The functional groups were tested by Fourier Transform Infrared Spectrometer .4Fe(CN)6 (3(HITP)2 MOFs mixed with Nafion was dropped on the polished GCE. Then, it was activated by the electrochemical method. The treated Ni-MOFs showed obvious reduction and oxidation peaks, which were sensitive to glucose and used as a non-enzymatic glucose sensor.The preparation of the Ni-MOFs glucose sensor was illustrated as 4Fe(CN)6 . After t−1 is the aromatic C-H stretching vibrations. The peak at 1453 and 1377 cm−1 was due to the C=C vibration of the aromatic polymer chain /Ni(III) to each other in the Ni-MOFs in the alkaline solution through the following reaction:The electrochemical responses of MOFs before and after activation were studied . It can The cyclic voltammogram of Ni-MOFs at different concentrations of KOH was also investigated . CompareCyclic voltammetry (CV) was used to study the glucose oxidation by treated Ni-MOFs. The CV curves showed that the peak current continually increased with the increasement of glucose concentration in the range of 0 to 10 mM with a linear calibration equation:Ipa is the anodic peak current and Cglucose is the concentration of glucose , linearly following the linear regression equation:The effects of scan rate were tested in the range of 10–100 mV s.1 M KOH . The anoThis suggests the diffusion of the glucose to the electrode surface was the limiting step of the electrochemical reaction.3(HITP)2-based glucose sensor with the reported literature 2 MOFs on GCE in an alkaline solution. Cyclic voltammetry technology was used to study the electrocatalytic oxidation of glucose on the surface of activated Ni-MOFs. The active MOFs displayed excellent electrocatalytic activity toward the glucose oxidation in 0.1 M KOH solution and showed a good linear relationship toward 0-10 mM. The diffusion of glucose to the electrode surface was the limiting speed for the electrochemical reaction. In addition, the MOFs sensor exhibited excellent selectivity toward glucose in the presence of AA and UC. Also, the electrode exhibited long-term stability.In conclusion, a non-enzymatic glucose sensor was developed through electrochemical activation of the NiThe conventional fabrication, high performance, excellent stability, and low cost of the activated conductive Ni-MOFs suggests its suitability as a reliable non-enzymatic glucose sensor.All datasets generated for this study are included in the article/ YC: conceptualization, methodology, validation, formal analysis, data curation, and writing original draft. YT: methodology, validation, investigation, visualization, and writing-review and editing. PZ: methodology, resources, and visualization. LD: methodology, writing-review and editing, and visualization. WC: conceptualization, methodology, validation, data curation, visualization, and writing-review and editing. CW: conceptualization, methodology, resources, writing-review and editing, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Interbody fusion is a common surgical technique for diseases of the lumbar spine. Biportal endoscopic-assisted lumbar interbody fusion (BE-LIF) is a novel minimally invasive technique that has a long learning curve, which can be a barrier for surgeons. Therefore, we analyzed the learning curve in terms of operative time and evaluated the outcomes of BE-LIF. A retrospective study of fifty-seven consecutive patients who underwent BE-LIF for degenerative lumbar disease by a single surgeon from January 2017 to December 2018 was performed. Fifty patients underwent a single-level procedure, and 7 underwent surgery at two levels. The mean follow-up period was 24 months . Total operative time, postoperative drainage volume, time to ambulation, and complications were analyzed. Clinical outcome was measured using the Oswestry Disability Index (ODI), Visual Analog Scale (VAS) score for back and leg pain, and modified Macnab criteria. The learning curve was evaluated by a nonparametric regression locally weighted scatterplot smoothing curve. Cases before the stable point on the curve were designated as group A, and those after the stable point were designated group B. Operative time decreased as the number of cases increased. A stable point was noticed on the 400th day and the 34th case after the first BE-LIF was performed. All cases showed improved ODI and VAS scores at the final follow-up. Overall mean operative time was 171.74 ± 35.1 min. Mean operative time was significantly lower in group B (139.7 ± 11.6 min) compared to group A (193.4 ± 28.3 min). Time to ambulation was significantly lower in group B compared to group A. VAS and ODI scores did not differ between the two groups. BE-LIF is an effective minimally invasive technique for lumbar degenerative disease. In our case series, this technique required approximately 34 cases to reach an adequate performance level. Harms first introduced the traditional open transforaminal lumbar interbody fusion (TLIF) in 1982 . Thus, mAmong the recently developed minimally invasive spinal surgical techniques, biportal endoscopic spinal surgery (BESS) has been applied for decompression as well as TLIF. Biportal endoscopic-assisted lumbar interbody fusion (BE-LIF) utilizes two separate percutaneous portals (viewing and working). It has several advantages over MI-TLIF due to its percutaneous approach, including minimization of retractor-induced muscle ischemia , 11. AltWe performed a retrospective study by reviewing the electronic medical records. This study was conducted after obtaining approval from the Institutional Review Board. Fifty-seven consecutive patients that underwent BE-LIF for lumbar degenerative disease by a single surgeon from January 2017 to December 2018 were enrolled. The indications of lumbar degenerative diseases requiring BE-LIF were degenerative spondylolisthesis and isthmic spondylolisthesis. We did not perform BE-LIF for revision or trauma cases. All patients received a minimum one-year follow-up, with the mean follow-up period of 24 months .The patient was positioned prone on a radiolucent operating table under general anesthesia. An arthroscopic imaging system for joint surgery was used for all cases. Initially, a true anteroposterior fluoroscopic view of the surgical site was obtained. For a left-sided approach, the proximal viewing portal and distal working portal were placed 1 cm above and below the surgical level, respectively. Both portal sites were used later as the entry points for percutaneous pedicle screws. For each portal, a 1 cm transverse skin incision was made; the fascial incision was made perpendicular to the skin incision to allow fluent outflow of irrigation fluid, which is essential for a clear intraoperative visual field. With fluoroscopic guidance, Cobb elevators were inserted percutaneously through the two portals. They were used to dissect the muscle away from the interlaminar space and proximal lamina to create a working space. Gravity-powered irrigation was used from a saline bag suspended at the height of 1.7 meters from the floor. To prevent obstruction of irrigation fluid outflow, a semitubular retractor was manually placed and held through the working portal. Soft tissue debridement and hemostasis were achieved using a radiofrequency coagulator. The subsequent steps resemble the MI-TLIF procedure. Proximal laminotomy and unilateral facetectomy were performed using a high-speed burr, Kerrison rongeur, and osteotome. Local autologous bone obtained during decompression was set aside for later use as bone graft. After the laminotomy and facetectomy, bilateral flavectomy was performed, and the shoulder portion of the traversing root and the disc space was exposed. Then, annulotomy and disc removal were performed. Tapered trial cages were serially inserted to expand the disc space adequately to allow insertion of the endoscope and instruments into the disc space. The endplates were prepared by gently removing the cartilage using a double-ended elevator while avoiding subchondral bone damage . The locOperative time, time to ambulation, length of stay, postoperative drainage amount, and complications were analyzed. The group before the stable point in terms of operative time was designated group A, and the group after the stable point was designated group B. A stable point was defined as the point that the line afterwards shows the smoothest trend. VAS and ODI data from before surgery and two weeks, two months, and after 1 year postoperatively were collected to evaluate lower back pain, leg pain, and disability. The modified Macnab criteria were checked on the final follow-up visit of each patient. For preoperative radiographic evaluation, anteroposterior, lateral, oblique, and flexion-extension lumbar plain radiographs, CT, and MRI were used. Interbody fusion was evaluated one year after surgery using lateral and flexion-extension plain radiographs. Radiographic fusion was assessed by two board-certified radiologists and defined as either formation of a trabecular bone bridge or <4 degrees of segmental motion on flexion-extension plain radiographs. The Bridwell grading system was used to classify fusion status  Figure .x-axis showing the date after the first case and the y-axis showing operative time. The chi-square test or two-sample t-test was used to compare clinical characteristics between the two groups. Clinical and radiographic results were compared using the two-sample t-test, chi-square test, Fisher's exact test, or repeated-measures ANOVA. SPSS software version 24.0  and MedCalc software version 19.1 were used for statistical analysis. p < 0.05 was considered significant.Data are reported as means ± standard deviation. A nonparametric regression locally weighted scatterplot smoothing curve was used to analyze the change of operative time, with the The mean patient age was 68.5 ± 9.4 years. Twenty-eight patients were male. Patient diagnoses included 46 cases of degenerative spondylolisthesis and 11 cases of isthmic spondylolisthesis. Fifty patients underwent single-level TLIF, and 7 underwent 2-level TLIF. The level of surgery in the single-level cases was L2-3 in 1, L3-4 in 5, L4-5 in 26, and L5-S1 in 18. Two-level TLIF was performed at L3-4 and L4-5 in 5 patients and L4-5 and L5-S1 in 2. Patient characteristics are summarized in p ≤ 0.001) (p = 0.007)  . The ODI≤ 0.001) . Unlike = 0.007) . While t= 0.007) . Modifie= 0.007) .th and 24th case, respectively, of group A and cage subsidence was found in group B. The postoperative spinal epidural hematoma was managed with revision surgery of surgical hematoma evacuation, which resulted in the improvement of back pain and neurologic symptoms.A complication occurred in 3 of the 57 patients (5.3%): 1 postoperative spinal epidural hematoma, 1 case of cage subsidence, and 1 case of transient paralysis. The postoperative spinal epidural hematoma and transient paralysis occurred in the 17Minimally invasive spinal surgery is effective in reducing soft tissue damage and recovery time compared to open surgery . NumerouTheoretically, since BE-LIF is performed percutaneously without retractors, muscle damage is presumably less compared to MI-TLIF. A previous study comparing BESS and microscopic surgery for unilateral laminotomy and bilateral decompression showed favorable results for BESS in terms of early postoperative clinical outcome. The authors assumed that such results were due to less tissue damage during BESS than open microscopic surgery . HoweverPreviously reported MI-TLIF complication rates were zero to 33% , 23. In In our case series, 55 of the 57 cases showed Bridwell grade I or II interbody fusion. Although the follow-up period was longer than the whole cohort, 2 cases showed grade III fusion in the early group. MI-TLIF has been reported to result in fusion rates ranging from 80% to 100% , 20, 27,However, BE-LIF has potential complications during surgery. For surgeons with insufficient experience, inadequate hemostasis, loss of orientation can prolong the operative time and may result in minor complications such as postoperative epidural hematoma, iatrogenic durotomy, or injury to the nerve root to major complications including nerve root palsy or intraspinal hematoma. Therefore, endoscopic training from cadaveric laboratories is recommended before attempting such technique , 30.There are several limitations to this study. First, this is a retrospective study investigating a heterogeneous disease entity. Second, only the duration of the entire operation rather than the individual operation steps such as laminotomy, discectomy, endplate preparation, or cage insertion was recorded. Third, the sample size is relatively small with a short follow-up period. Finally, there is a performance bias in this study that the operating surgeon has been performing BESS for decompression or discectomy for the last 1 year before starting BE-LIF. The required cases for adequate performance cannot be generalized since the actual learning curve can vary among individuals.BE-LIF is an effective minimally invasive technique for lumbar degenerative disease. It can result in significant clinical improvement with a low risk of complications, which seems to be comparable to the results of MI-TLIF. In our case series, 34 cases were required to reach a constant and adequate performance level.


Deceased patients were split into two groups according to age at the time of death: n = 26 adult patients aged below 60 and n = 85 elderly patients over 60. With the use of HLA class I genotypes, we developed a risk score (RS) which was associated with the ability to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides by the HLA class I molecule set of an individual. The resulting RS was significantly higher in the group of deceased adults compared to elderly adults . In particular, presence of HLA-A*01:01 allele was associated with high risk, while HLA-A*02:01 and HLA-A*03:01 mainly contributed to low risk. The analysis of patients with homozygosity strongly highlighted these results: homozygosity by HLA-A*01:01 accompanied early deaths, while only one HLA-A*02:01 homozygote died before 60 years of age. Application of the constructed RS model to an independent Spanish patients cohort (n = 45) revealed that the score was also associated with the severity of the disease. The obtained results suggest the important role of HLA class I peptide presentation in the development of a specific immune response to COVID-19.Human leukocyte antigen (HLA) class I molecules play a crucial role in the development of a specific immune response to viral infections by presenting viral peptides at the cell surface where they will be further recognized by T cells. In the present manuscript, we explored whether HLA class I genotypes can be associated with the critical course of Coronavirus Disease-19 by searching possible connections between genotypes of deceased patients and their age at death. HLA-A, HLA-B, and HLA-C genotypes of  In response to the interaction, the CD8+ T lymphocyte activates and starts to divide; in 5–7 days, a population of virus-specific cytotoxic CD8+ T lymphocytes capable of destroying infected cells using perforins and serine proteases is formed (+ T cells activation in the immune response to COVID-19 has been recently studied in a cohort of patients who had mild disease (Human leukocyte antigen (HLA) class I molecules are one of the key mediators of the first links in the development of a specific immune response to Coronavirus Disease-19 (COVID-19) infection. Right after entering the cell, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces the translation of its proteins. Some of these proteins enter the proteasomes of the infected cell, become cleaved to peptides the length of 8–12 amino acid residues, and bind to HLA class I receptors. After binding, the complex consisting of the HLA class I molecule and the peptide is transferred to the surface of the infected cell, where it can interact with the T cell receptor of CD8 disease .There are three main types of HLA class I receptors: HLA-A, HLA-B, and HLA-C. Receptors of every type are present in two variants inherited from parents. There exist dozens of variants of each allele of HLA-I receptors; every allele has an individual ability to recognize various foreign proteins. The distribution of alleles is population/country specific .*07:03 , and searched for putative associations between genotypes and age at death. Since the total number of distinct HLA class I genotypes is too high to perform frequency-based analysis, we assigned scores to each allele based on capability of presenting SARS-CoV-2 peptides. The obtained scores allowed us to make a valid statistical comparison between HLA genotypes in groups of deceased adults , elderly adults , and the control. Special attention was paid to “extreme” cases formed by homozygous individuals by some HLA genes. Additionally, we assessed the contribution of each viral protein to the constructed risk model.In the present study, we explored whether HLA class I genotypes can be a factor contributing to the critical course of COVID-19. For that, we performed HLA genotyping for post-mortem. Patients were divided into two groups according to their age at death: adults  and elderly adults .There were 111 patients infected with COVID-19 enrolled in O.M. Filatov City Clinical Hospital,  who died between May and July 2020. All patients had at least one positive test result for SARS-CoV-2 by reverse transcription PCR (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavage. Patients with pathologies that led to greater morbidity or who had additional immunosuppression  were not included in the study. Blood (2 ml) was collected by the medical practitioner from the right ventricle in an ethylenediaminetetraacetic acid (EDTA) vial The control group of 428 volunteers was established with the use of electronic HLA genotype records of the Federal Register of Bone Marrow Donors . All patients or their next of kin gave informed consent for participation in the study.The study protocol was reviewed and approved by the Local Ethics Committee at the Pirogov Russian National Research Medical University ; the study was conducted in accordance with the Declaration of Helsinki.Genomic DNA was isolated from frozen collected anticoagulated whole blood samples using the QIAamp DNA Blood Mini Kit on the automatic workstation QIACube . HLA-A, HLA-B, and HLA-C genes were sequenced with the MiSeq platform  through exons 2–4 in both directions using reagent kit HLA-Expert  and annotated using the database of the human major histocompatibility complex (MHC) sequences IMGT/HLA v3.41.0 . Processn = 79) were obtained from GISAID , we compared the obtained alignment with the mentioned reference. Two protein sequences differed in four positions (0.04%) located within the NSP12, N, and S viral proteins. Such differences can indeed lead to functional diversity of analyzed proteins, but will be totally negligible for further analysis.We applied the procedure described by Nguen et al.  to the cn = 15,314) and HLA alleles present in our cohorts of deceased and control patients (n = 107). Peptides with a weak binding affinity to all considered alleles were discarded . For the remaining 6,548 peptides, all affinities were inverted, multiplied by 500 and log10-transformed. Thus, the resulting score was equal to zero for peptides with a weak binding affinity threshold (500 nM) and equal to one for a high binding affinity (50 nM). Raw and processed matrices are presented in Binding affinities were predicted using netMHCpan v4.1  for all U test. The Benjamini-Hochberg procedure was used to perform multiple testing corrections. Principal component analysis was conducted with the scikit-learn Python module . The following functions from the scipy.stats Python module  were usen module . Permutatplotlib .n = 111 deceased patients with confirmed COVID-19  and the control group consisting of volunteers (n = 428). Deceased patients were divided into two groups: adults (age at death ≤ 60 years) and elderly adults (age at death over 60 years). Demographic and clinical data of these cohorts are summarized in p = 1.89 × 10−3). Other cardiovascular diseases like coronary artery disease and heart failure were also more frequent in the group of elderly individuals which, however, was not statistically significant. Interestingly, arterial hypertension was diagnosed in 11.5% of adult patients and 24.7% of older adults, which was generally less than populational level in Russia (about 50%) (We performed HLA class I genotyping for out 50%) . Percentout 50%) .p-values were equal to 1). However, few of them were differentially enriched if no multiple testing corrections were applied  fell in different clusters, while for HLA-B and HLA-C, some major alleles were grouped together .As a result, we obtained a matrix containing affinity values for 6,548 peptides and 107 alleles of genes from major HLA class I. To establish a positive relationship between values from the matrix and binding potential, all affinities were inverted and scaled by a value of 500 nM . Simultaneous hierarchical clustering was applied to identify groups of similar peptides and HLA-A, HLA-B, and HLA-C alleles . As can *06:02, HLA-C*07:01, and HLA-C*07:02. From the analysis of the contingency table of allele pairs in the control group , HLA-C*07:01 is linked to HLA-A*01:01 (p = 2.97 × 10−7) and HLA-B*08:01 (p = 3.15 × 10−14), while HLA-C*07:02 is coupled with HLA-A*03:01 (p = 9.66 × 10−4) and HLA-B*07:02 (p = 2.72 × 10−26). Interestingly, such linked alleles can have different peptide binding patterns . In this framework 6,548-element affinity vectors are replaced by the most informative linear combinations of their components. For HLA-A and HLA-C, we found four principal components (PCs), each of which explained at least 5% of data variance, while for HLA-B, the number of essential components was equal to five . Signs oU test. This list included the second and third PCs of HLA-A, and the fourth PC of HLA-C, while no PCs of HLA-B separated the analyzed groups significantly. As an aggregate risk score (RS), we considered the sum of these three components . The obtained score also significantly separated groups with p = 3.48 × 10−3 (U test) and AUC ROC equal to 0.68 (permutation test p = 3.10 × 10−3), see U test p = 3.31 × 10−3), while the difference between the elderly and control groups was not (p = 0.283).For each individual, HLA class I gene, and PC, we summed PC values associated with two corresponding alleles. After that, we analyzed differences of obtained scores in adult and elderly patients who died from COVID-19. Three of the resulting PCs demonstrated statistically significant differences according to the Mann-Whitney p-value for Fisher's exact test applied to the number of adult and elderly patients in the whole cohort and in the low/high risk groups, respectively. Interestingly, such partitioning led to significant separation of adult patients both from the elderly and control subjects in the low and high risk groups, while no significance was found within the middle group . Then, the set of the most RS contributing peptides was composed by taking the top 5% of peptides from the corresponding distribution. The results of the procedure are summarized in n = 20), moderate (n = 20), and mild (n = 5) SARS-CoV-2 infection. The RS model was applied to the data with no re-tuning of coefficients, and the same PCA weights were used for cohort-specific alleles. As a results, we found statistically significant dominance of RSs in patients with severe symptoms compared to moderate (U test p = 0.0157) and mild (p = 0.0161) patients, while, despite the matching direction, no statistical significance was found for the comparison of patients with moderate and mild infection (p = 0.0926), possibly due to the low sample size . The distribution of homozygous individual by HLA-A*01:01 among the groups of deceased patients and the control group proved its negative role. It turned out that the distribution in the deceased group  leads to two statistically significant differences: p-value of Fisher's exact test comparing the adults group with the elderly group equals 3.10 × 10−3, and p-value of the test comparing the adults group and the control group equals 0.0104 (8 out of 428 members of the control group were homozygous by HLA-A*01:01). However, the difference between the elderly group and the control group is statistically insignificant (p = 0.155). Interestingly, there were no other statistically significant differences in the distribution of homozygosity between the groups.When analyzing the high RS group, we noticed that more than half (five out of eight) deceased patients containing HLA-AU test p = 6.45 × 10−3, p = 6.03 × 10−3) and the control group . Difference between the elderly and control groups was not statistically significant (p = 0.448).Generally, the average age of death for patients homozygous by any allele was significantly less compared to heterozygous ones (Mann-Whitney *02:01 (six cases) and HLA-A*03:01 (two cases) alleles were associated with low risk. These assignments were in agreement with age of death: only one patient homozygous by HLA-A*02:01 had not passed the 60 years age of death threshold. Thus, homozygosity by HLA class I genes is generally associated with poor prognosis except for some alleles like HLA-A*02:01 and HLA-A*03:01 with “relevant” peptide-binding profiles.However, the low risk group also contained homozygous individuals: all homozygosity cases by HLA-AIn the current study, we presented the characteristics of a large cohort of deceased patients with COVID-19 from O.M. Filatov City Clinical Hospital in Moscow, Russia. The clinical characteristics of these patients indicated that the HLA genotype, age, and underlying diseases were the most important risk factors for death. In our population, the median age of deceased patients was 73.0 years. Three previous studies reported an average age in non-survivors to be 78.0, 65.8, and 70.7 years old, respectively –28. Our The majority of deceased patients at age <60 were men (61.5%), while the populational level for this age category in Russia was 48% . Such imOnly 18 deceased elderly patients (21.2%) had no comorbidities. A number of previous studies mentioned a high percentage of comorbidities in a group of patients with the severe course of COVID-19 , 30, 31.*07:29 and HLA-B*15:27 , we assigned a numerical value to each allele associated with the aggregate binding affinity of viral peptides to the corresponding receptor. The obtained RS separated adult patients who died due to COVID-19 from both the elderly subjects and the control group with a statistical significance. A conceptually similar technique was used by Iturrieta-Zuazo et al.: allele score was calculated as a number of tightly binding viral peptides (affinity <50 nM) . However*02:01 and HLA-A*03:01 were tightly associated with low risk while HLA-A*01:01 contributed to the high risk group. Connection of HLA-A*01:01 and HLA-A*02:01 alleles with COVID-19 morbidity and mortality was already mentioned in the existing literature, namely, frequency of HLA-A*01:01 in Italian regions positively correlated both with the number of COVID-19 cases and deaths, while significant negative correlation was observed for HLA-A*02:01  vs. four out of 100 infected (4%) patients (Fisher's exact test p = 1.40 × 10−3). Thus, presentation of “important” viral peptides with doubled intensity can enhance immune response showing that HLA class I homozygosity can act like a double-edged sword.An interesting illustration for the role of HLA-A to AIDS . On the -authors : none ou+ T cell epitopes by taking the top 1% of peptides according to their binding affinity to 12 common HLA class I alleles . The patients/participants provided their written informed consent to participate in this study.MS, SN, and AT: conceptualization and validation. MS, SN, TJ, AG, IG, VV, and AT: methodology, investigation, and writing (review and editing). SN and AG: software and formal analysis. MS and SN: writing . All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study determined whether antioxidant supplementation is a viable complement to exercise regimens in improving cognitive and motor performance in a mouse model of Alzheimer’s disease risk. Starting at 12 months of age, separate groups of male and female mice expressing human Apolipoprotein E3 (GFAP-ApoE3) or E4 (GFAP-ApoE4) were fed either a control diet or a diet supplemented with vitamins E and C. The mice were further separated into a sedentary group or a group that followed a daily exercise regimen. After 8 weeks on the treatments, the mice were administered a battery of functional tests including tests to measure reflex and motor, cognitive, and affective function while remaining on their treatment. Subsequently, plasma inflammatory markers and catalase activity in brain regions were measured. Overall, the GFAP-ApoE4 mice exhibited poorer motor function and spatial learning and memory. The treatments improved balance, learning, and cognitive flexibility in the GFAP-ApoE3 mice and overall the GFAP-ApoE4 mice were not responsive. The addition of antioxidants to supplement a training regimen only provided further benefits to the active avoidance task, and there was no antagonistic interaction between the two interventions. These outcomes are indicative that there is a window of opportunity for treatment and that genotype plays an important role in response to interventions. Apolipoprotein E4 (ApoE4) is the most prevalent genetic risk factor for late-onset Alzheimer’s disease (AD) . In miceOxidative stress has been associated with cognitive and motor declines and has been suggested as a major contributor to AD pathology. Oxidative stress has also been involved in vascular cognitive impairment, a risk for dementia . The braAnother well-marketed healthy lifestyle modification is exercise. Physical activity has been associated with a reduced risk of AD , delayedBoth lines of therapy improved behavioral outcomes associated with AD, and seemed to at least partially involve oxidative stress as part of their mechanism of action. Therefore, it can be hypothesized that combining antioxidants with exercise training will lead to an additive beneficial effect, reducing impairments. While some reports have determined that there can be such a positive interaction ,27,28, oThe goals of the current study were to determine (1) cognitive, motor, and anxiety phenotypes of middle-aged GFAP-ApoE3 and E4 male and female mice; (2) whether antioxidant intake or exercise training leads to functional improvements; (3) whether the combination of antioxidant and exercise yields an additive beneficial effect; (4) the involvement of oxidative stress and inflammation in behavioral outcomes. Our hypothesis was that moderate exercise and antioxidants would lead to additional beneficial effects when compared to each intervention alone, which would be exacerbated in the ApoE4 genotype. The outcomes are important in deciding the need for antioxidant supplementation in exercising individuals, and as a guiding parameter in genotype-based experiments.APOE3)37Hol APOEtm1Unc/J) and GFAP-ApoE4 (B6.Cg-Tg(GFAP-APOE4)1Hol APOEtm1Unc/J) mice were obtained from The Jackson Laboratory   at 2 months. These mice express the human apolipoprotein E3 or E4 under the control of the glial fibrillary acidic protein (GFAP) promoter and do not express endogenous ApoE. The mice were group housed in standard housing conditions and received ad libitum access to food and water at an ambient temperature, under a 12-h light/dark cycle starting at 06:00.All animal use protocols were approved by the Institutional Animal Care and Use Committee at UNT HSC . Groups of male and female GFAP-ApoE3  was modified by adding ascorbic acid (1.65 mg/g diet) and α-tocopheryl acetate (1.12 IU/g diet) (#5SH0). Using treadmills, the mice were progressively introduced to a moderate exercise regimen . The training was increased in time and speed to reach a maximal exercise of 1 h over 12 days . Compliance was achieved by a transient 0.29 mA electric foot shock to the feet. The number of shocks were tallied, and the paired control mice received the same number of shocks/training day.When the mice were 12 months old, they were randomly allotted to: (1) sedentary + control diet (Sed-Con), (2) sedentary + control diet supplemented with vitamins E and C (Sed-EC), (3) exercise + control diet (Ex-Con), (4) exercise + control diet supplemented with vitamins E and C (Ex-EC). The diets were obtained from TestDiet . The control diet (LabDietThe mice were weighed weekly and food intake (average of five consecutive days) was recorded the week before the start of behavioral testing.For 16 weeks, the mice were on their respective treatments, including 8 weeks prior to the behavioral assessments. The behavioral tasks were described in details previously ,31.The position of a mouse in the maze  was determined by a tracking system . Under dim light (60 W) and for 5 min, each mouse was left to explore. Time in open arms was used to measure anxiety levels.Each mouse was left to explore an acrylic test chamber (40.5 cm × 40.5 cm × 30.5 cm) under dim light and with background noise (80 dB) for 16 min. Their activity was recorded using a Digiscan apparatus . Their horizontal, vertical, and spatial movements were detected by the photocells and processed by a software program.A motor-driven cylinder rotating at increasing speed was used to measure motor learning and running performance . The mice were given two training sessions per day ), which continued until improvements failed over three consecutive sessions. The average of latency to fall for the four trials in each session was used for motor learning and for the final session when stable performance was achieved.Walk initiation: record the latency to move one body length immediately after being placed on a smooth surface. Alley turn: record the latency to make a full turn in a dead-end alley. Negative geotaxis: record the latency to turn 180 ° in either direction when placed facing downward on a 45 ° tilted grid. Wire suspension: record the latency to grasp wire with hindpaws after being suspended from wire by front paws and the latency to fall . The mice received four sessions (one/day) and latencies were averaged across the four sessions. A maximum of 60 s for each test was used.Each mouse was placed on one of four bridges  and latency to fall (maximum of 60 s) was recorded. Each bridge was presented three times and latency to fall was averaged for each bridge and across bridges.Spatial learning and memory were measured using a modified Morris water maze test. Prior to any testing, the mice received pre-training using a covered straight alley to teach the mice to swim and climb onto a platform. Following this, the mice were given a maximum of 90 s to find a hidden platform from different starting positions . Their performance was tracked via Any-maze software . Path length and swim speed were used as measures of performance.Using a T-maze apparatus set on a grid floor set to deliver 0.69 mA scrambled shock, we tested the mice for learning and cognitive flexibility using a discriminated avoidance task. During the information trial (first one), a shock is administered when the mouse enters their preference arm (first one) and allowed to escape the shock by running to the opposite arm (correct arm). Thereafter, the initiation of shock was 5 s upon start door opening or upon entry into an incorrect arm until the mouse entered the correct arm or a maximum of 60 s. After 10 s in the correct arm, the mouse was placed in a holding cage for 1 min (ITI). Training continued until a criterion of correct avoidance was reached . The reversal sessions followed the same training and switching of the correct arm. Learning ability was considered inversely proportional to the number of trials required to reach the avoidance criterion.After behavioral testing, blood was collected via tail bleed prior to euthanization . Brains were harvested and dissected into brain regions that were saved at −80 °C .2O2. The production of the resulting formaldehyde was measured colorimetrically with Purpald  at 540 nm.The activity of the antioxidative enzyme, catalase, was evaluated using the Catalase Assay Kit from Cayman Chemical . Methanol was reacted with catalase in the presence of for the optimal concentration of H® analysis software . While the panel measures more than these four markers, the others were out of range based on the manual and literature searches. Therefore, we did not include them in our analyses.Plasma levels for IL-10, IL-6, and TNFα were measured using an MSD Inflammatory Panel Kit from Meso Scale Diagnostics, LLC, Rockville, MD, USA) (10-plex Proinflammatory Panel 1 (mouse) kit (cat # C4048-1) and analyzed by MSD DISCOVERY WORKBENCHn). Planned individual comparisons between different sexes, genotypes, and treatments were performed using single degree-of-freedom F-tests involving the error term from the overall ANOVA. Some behavioral performances and catalase activity were also considered in four-way analyses with session or brain region as the repeated measure. The software used for the analyses was Systat 13  and p was set< 0.05.The functional performance of the mice on the behavioral tests, as well as the biochemical measurements, were assessed using three-way analyses of variance (ANOVA) with sex, genotype, and treatment as between-group factors . By week 7, most mice lost 3–7% of their body weight, and the only significant differences were between E3 and E4 Sed-Con females, and between Sed-Con and Sed-Aox in the GFAP-ApoE4 females . By week 12, the loss was more pronounced (up to 13%), and the only significant differences were Ex-Aox GFAP-ApoE4 females losing less weight than Sed-Con (p < 0.05), while in males, the Ex-Aox groups lost the most weight (p < 0.05 only for GFAP-ApoE3). Food intake was not significantly affected by sex, strain or treatment  (not shown).Percent changes in body weight from the week before behavioral testing started and at the end of the study are presented in p < 0.001) and no effect of strain (p = 0.366). Treatments did not affect performance , with the exception of Sed-Aox group spending more time than the Sed-Con group in the open arms (p = 0.046). Overall, the GFAP-ApoE3 mice were more active than the GFAP-ApoE4 mice, and some treatments affected the activity of females . These observations were supported by the significant main effects of strain and treatment and a sex × strain × treatment interaction. .The performance of the mice in this test measuring affective function was analyzed and is presented in p < 0.001) and no effect for treatment, sex or any interactions between the factors . GFAP-ApoE4 mice also spent 20% less time rearing than the GFAP-ApoE3, supported by a main effect of strain (p = 0.001). Most treatments had no effect on the rearing activity of the mice with the exception of Sed-Aox, which increased it (p = 0.046).Horizontal distance and rearing activity were analyzed . GFAP-App < 0.003). A main effect of treatment (p = 0.014) was due to Ex-Con and Ex-Aox GFAP-ApoE3 mice exhibiting better performance in males . The Ex-Con GFAP-ApoE4 group also seemed to have a better performance (p = 0.051) which was not seen in the Ex-Aox group. During the plateau phase, there was only an effect of strain, in which GFAP-ApoE4 mice had a poorer performance than the GFAP-ApoE3 mice (p < 0.001).The data were averaged as learning (sessions 1–4) and plateau (sessions 5–7) performance . During p = 0.019). There was no effect of treatment, sex, or interactions between any of the factors . Alley-turning was affected differentially by the treatments depending on sex and genotype . GFAP-ApoE4 females took longer latencies than the GFAP-ApoE3 females, while there was no difference in males. In GFAP-ApoE4 females, Sed-Aox took longer to turn, while in GFAP-ApoE3, the Ex-Aox males had the shortest latency. For negative geotaxis, males took longer latencies to turn, as well as GFAP-ApoE4 mice. The Sed-Aox mice had shorter latencies in the GFAP-ApoE3 groups . Latency to tread was affected by strain (p = 0.01), due mainly to treatments affecting each genotype differently . There was no effect of treatment or interactions between any of the factors .Performance was averaged over four sessions and is presented in p > 0.13). In session 2, females performed better than males leading to a main effect of strain. While there was no main effect of treatment, exercised groups had higher latencies in males regardless of strain but it only reached significance for the Ex-Aox GFAP-ApoE3 mice (p = 0.029). In session 3, a main effect of treatment was obtained (p = 0.03) due to all treatments in male GFAP-ApoE3 mice. In session 4, treatments, especially exercise, were associated with increased latencies in the GFAP-ApoE3 but not GFAP-ApoE4 groups. GFAP-ApoE4 females performed better than their male counterparts, driving the main effects of sex and strain .The latency to fall was analyzed for each bridge and is presented in p = 0.044). During the learning phase, female mice had higher path lengths than the males . Treated GFAP-ApoE4 females had lower latencies than the sex- and strain-matched Sed-Con group (p = 0.064), however, it only reached significance with the Ex-Aox mice. Maximum performance was impaired in the GFAP-ApoE4 males, and was reversed by all treatments. However, an ANOVA did not reach significance for strain (p = 0.098) or strain x treatment (p = 0.096). Females swam slower than males, GFAP-ApoE4 mice seemed slightly faster than GFAP-ApoE3 mice, and none of the treatments affected the swimming speed (not shown). ANOVA yielded a main effect of sex , but the effect of strain did not reach significance (p = 0.057).The performance of the mice was separated into initial, learning phase, and maximum performances . During p = 0.005). Sed-Aox (p = 0.035) and Ex-Con (p = 0.052) groups took fewer trials to reach criterion in the GFAP-ApoE3 males and there was no significant effect in the GFAP-ApoE4 mice. In females, Ex-Con (p = 0.058) and Ex-Aox (p = 0.019) performed better than the Sed-Con group in the GFAP-ApoE3 group, while only Ex-Aox (p = 0.018) took fewer trials than the controls in the GFAP-ApoE4 group.Performance during acquisition and reversal is presented in p = 0.007), mainly in the GFAP-ApoE3 mice, but this observation was not supported by an interaction between strain and treatment (p = 0.23). There was no significant effect of any of the treatments in the GFAP-ApoE4 mice. In the GFAP-ApoE3 group, all treated mice took fewer trials compared to Sed-Con in females and Ex-Aox was the only significantly different group in males (p = 0.029).During reversal, treatments improved performance . Overall, the activity was ranked as follows: hippocampus > cerebellum, midbrain > cortex, supported by a main effect of region (p < 0.001). There was an overall effect of treatment (p = 0.003) which was not region-dependent , but no effect of genotype . The effect of treatment was due mostly to effects of Ex-Aox in all regions (not significant for cerebellum).Catalase activity was measured in the cerebral cortex, hippocampus, cerebellum, and midbrain and is presented in p = 0.055). The female groups that exercised had lower levels of IL6 than the control groups. There was no effect of sex, strain, or treatment on TNFα levels , though the Ex-Aox GFAP-ApoE4 females had higher levels than the controls (p < 0.05).Levels of IL6, TNFα, and IL10 were measured in the plasma and are reported in The main findings of this study were: (1) there were strain differences for most motor functions, but no major differences for strength, balance, or cognition; (2) there were strain and test-dependent differences in response to the treatments: GFAP-ApoE3 mice were responsive on bridge and active avoidance, while GFAP-ApoE3 mice were on spatial learning and memory; (4) the most effective treatment was exercise, and no major additive or antagonistic effects were observed with antioxidant intake.The current study provided a comprehensive phenotype of this mouse model, and its response to non-conventional therapeutic interventions. Anxiety, the most common non-cognitive symptom of AD  and assoAPOE4 mice performed poorly [During its early phase, AD is often associated with motor function impairments. Pathological changes in the motor cortex, striatum, cerebellum, or substantia nigra might be responsible for motor decline in AD ,41. The d poorly . Repetitd poorly . Exercisd poorly . The lacd poorly , though d poorly .Different mouse models expressing human ApoE4 exhibited poor spatial learning and memory ,38,42. OAD symptoms start with the loss of non-spatial short-term/working memory . While wContrary to expectation, catalase activity was reduced in brain regions in mice that were exercised, especially the ones combined with antioxidants. Reports on the effects of exercise on catalase activity in the brain are conflicting: from no effect  to up-reThe presence of ApoE4 is associated with an increased risk of developing late-onset AD, however, clinically, it does not seem to always be true with AD patients not carrying the ε4 and vice versa, suggesting a more complicated picture and gene involvement in AD etiology . FurtherWhile it remains under debate that these non-pharmacological therapies are effective in reducing neurodegeneration, it is possible that they affect the vascular contribution to these diseases. By managing and controlling the vascular risk factor via a healthy lifestyle, such as exercising and improving one’s diet, it is likely to reduce vascular cognitive impairments and reduce the advance to neurodegenerative diseases and dementias . These nOur study has several limitations: (1) the model used may not be generalizable to all AD patients, as it focused on the ApoE genotype and on a glial model of ApoE expression; (2) the limited oxidative stress measurements, as such glutathione levels or protein damage measurements would have strengthened the role of oxidative stress in the study ; (3) theExercise was the most consistent treatment effective at improving motor and cognitive function, and the addition of antioxidants did not lead to major additive or antagonistic effects. ApoE4 mice were less responsive to the treatments than the ApoE3 mice, suggesting a genotype-dependent response to interventions. Therefore, factors such as sex, age, genotype, and chosen tests need to be carefully incorporated into preclinical studies of interventions to improve brain function during aging or neurodegenerative diseases.


The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression. The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases. This study defines an endocytic trafficking pathway regulated by FCHSD2 and Rab7 that controls receptor tyrosine kinase expression and oncogenic signal transduction, opening the possibility of novel therapeutic strategies for cancer. Non-small-cell lung cancer (NSCLC) is the leading cause of death among cancers worldwide . TransfoCME is the major endocytic pathway that determines the rates of internalization of plasma membrane receptors, regulates their expression on the cell surface, and controls their downstream signaling activities –7. We prDrosophila ortholog, Nervous Wreck (Nwk), suppresses bone morphogenetic protein (BMP) signaling, but by regulating endosomal recycling at the synapse  and pMD2G) were co-transfected into HEK293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions for virus production. The virus-conditioned media were used to infect target cells in the presence of 10 μg/ml polybrene (MilliporeSigma). After infection for 48 h, the cells were grown in media containing puromycin (2.5 μg/ml) for 2 wk before experiments.Cells cultured in each well of a 6-well plate at 80% confluency were washed 3 times with PBS and harvested/resuspended in 150–200 μl of 2× Laemmli buffer . The cell lysate was boiled for 10 min and loaded onto an SDS gel. After transferring to a nitrocellulose membrane (Bio-Rad), membranes were blocked with 5% milk in TBST buffer and were probed with antibodies diluted in 5% BSA in TBST buffer against the following proteins: FCHSD2 , Rab11 , Rab7 , p-EGFR Y1068 , EGFR , p-MET Y1234/1235 , MET , p-Akt S473 , Akt , p-ERK1/2 T202/Y204 , ERK1/2 , p-STAT3 Y705 , STAT3 , p-c-Jun S63 , c-Jun , p-ETS1 T38 , ETS1 , β-Actin , Histone-H3  and vinculin . Horseradish peroxidase (HRP)-conjugated secondary antibodies  were used according to the manufacturers’ instructions. Quantitative analysis was performed by using ImageJ software .5) were seeded in each well of a 6-well plate containing RPMI 1640 with 10% FBS. Eight hours after seeding, cells were washed 3 times with PBS and starved in RPMI 1640 without FBS for 16 h. The cells then were untreated or treated with 100 ng/ml of EGF or HGF in the presence of cycloheximide (40 μg/ml) for the indicated times. After the stimulation, cells were washed 3 times with PBS and harvested/resuspended in 150–200 μl of 2× Laemmli buffer, and the cell lysates were subjected to western blotting and image analysis as described earlier.For EGF- or HGF-induced degradation of EGFR or MET, after siRNA transfection, the cells  for either a 10- or 30-min pulse at 37 °C. Cells were then immediately cooled down (4 °C) to stop internalization. The remaining surface-bound biotinylated Tfn was cleaved by incubation with 10 mM Tris (2-carboxyethyl) phosphine (TCEP) in assay buffer for 30 min at 4 °C. The surface-bound biotinylated EGF was removed by acid wash  at 4 °C. For TfnR recycling assays using ERK1/2 inhibitors (SCH772984), cells were incubated in the absence or presence of 10 μM of SCH772984 in the assay buffer containing 10 mM TCEP for 30 min at 4 °C before recycling assays were performed in the continued absence or presence of the inhibitor. Cells were washed with cold PBS4+ buffer and then incubated in PBS4+ containing 2 mg/ml of holo-Tfn  or 100 ng/ml of EGF and 10 mM of TCEP at 37 °C for the indicated times. The recycled biotinylated Tfn or biotinylated EGF was removed from the cells by the acid wash step. Cells were then washed with cold PBS and then fixed in 4% paraformaldehyde (PFA)  in PBS for 30 min and further permeabilized with 0.1% Triton X-100/PBS for 10 min. Remaining intracellular biotinylated ligands were assessed by streptavidin-POD  in Q-PBS, which contains 0.2% BSA , 0.001% saponin (MilliporeSigma), and 0.01% glycine (MilliporeSigma). The reaction was further developed with OPD (MilliporeSigma) and then stopped by addition of 50 μl of 5 M of H2SO4. The absorbance was read at 490 nm . The decrease in intracellular biotinylated ligands (recycling) was represented as the percentage of the total internal pool of ligand internalized. Well-to-well variability in cell number was accounted for by normalizing the reading at 490 nm with a BCA readout at 562 nm.TfnR recycling assays were performed using biotinylated Tfn, which was conjugated at a 7:1 molar ratio with the cleavable EZ-Link Sulfo-NHS-SS-Biotin  according to the manufacturer’s instructions. For EGFR recycling, non-cleavable biotinylated EGF  was used for assays. TfnR and EGFR recycling assays were performed as previously described . In brieAfter siRNA transfection, the cells were washed 3 times with PBS and then starved in RPMI 1640 medium without FBS for 30 min at 37 °C. Cells were incubated with 20 ng/ml EGF or 1 μg/ml HGF in RPMI 1640 medium for 30 min at 4 °C, washed with cold PBS, and then incubated in pre-warmed RPMI 1640 medium at 37 °C for the indicated times. Cells were then washed with ice-cold PBS to stop chase, fixed with 4% (w/v) PFA for 30 min at 37 °C, and permeabilized using 0.05% saponin (w/v) (MilliporeSigma) for 10 min. Cells were blocked with Q-PBS and probed with antibodies against the following proteins: p-EGFR Y1068 , MET , EEA1 , Rab11 , and LAMP1 , according to the manufacturers’ instructions. AlexaFluor-conjugated secondary antibodies  were used according to the manufacturers’ instructions. Fixed cells were mounted in PBS and imaged using a 60×, 1.49 NA APO objective  mounted on a Nikon Ti-Eclipse inverted microscope coupled to an Andor Diskovery Spinning disk confocal/Borealis widefield illuminator equipped with an additional 1.8× tube lens . The pinhole size was 50 μm. The percentages of colocalizations were determined using ImageJ software .GAPDH.Cell samples were homogenized, and total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) and treated with DNase I  according to the manufacturer’s instructions. Equal microgram volumes of RNA were reverse transcribed for each experimental condition by the use of a high-capacity cDNA reverse transfection kit  with oligo dT . qPCR was carried out using SYBR green reagents in technical duplicates (Thermo Fisher Scientific) on an ABI QuantStudio 5 Real-Time PCR System; primer sequence information is in After siRNA transfection, the cells were subjected to fractionation using the Nuclear/Cytosol Fractionation Kit  according to the manufacturer’s instructions.After siRNA transfection, the cells were collected in ice-cold PBS containing protease and phosphatase inhibitor cocktails . The cells were used to measure Rab7 activation using the Rab7 Activation Assay Kit  according to the manufacturer’s instructions.P value was calculated by logrank test [NSCLC patient survival data were downloaded from the Kaplan-Meier plotter database . Analysiank test .The human lung adenocarcinoma tissues were from US Biomax, Rockville, MD (#LC641) and the UT Southwestern Tissue Resource, a shared resource at the Simmons Comprehensive Cancer Center, Dallas, TX. The tumors were classified according to the American Joint Committee on Cancer (AJCC) TNM system. The immunohistochemical staining of FCHSD2  was optimized and performed by the core facility. The immunohistochemical images were analyzed using the IHC Profiler, ImageJ software (NIH) as previously described  to classt tests as indicated. P < 0.05 was considered significant.GraphPad Prism  was used to perform statistical analyses. Data were assessed by two-tailed, unpaired Student S1 Data(XLSX)Click here for additional data file.S1 Raw ImagesThe loading order, experimental samples, and molecular weight markers are indicated. The lanes used in the final figure are marked with a red box.(PDF)Click here for additional data file.S1 Fign = 3). Two-tailed Student t tests were used to assess statistical significance versus siCtrl. *P < 0.05, **P < 0.005. The underlying data for this figure can be found in Endocytic recycling of TfnR was measured in control or FCHSD2 siRNA-treated HCC4017 and ARPE-19 cells in the absence or presence of the ERK1/2 inhibitor SCH772984 (10 μM). Cells were pulsed for 30 min with 10 μg/ml biotinylated Tfn, stripped, and reincubated at 37 °C for the indicated times before measuring the remaining intracellular Tfn. Percentage of recycled biotinylated Tfn was calculated relative to the initial loading. Data represent mean ± SEM ((TIF)Click here for additional data file.S2 Fign = 3). Two-tailed Student t tests were used to assess statistical significance. *P < 0.05, **P < 0.005, ***P < 0.0005. (C) Representative confocal images of pEGFR and LAMP1 immunofluorescence staining in control or FCHSD2 siRNA-treated H1975 cells. Cells were incubated with 20 ng/ml EGF for 30 min at 4 °C, washed, and re-incubated at 37 °C for the indicated times. Scale bar, 12.5 μm. (D) Colocalization of pEGFR and LAMP1 immunofluorescence staining in the cells as described in (C). Data were obtained from at least 40 cells in total/condition and represent mean ± SEM. Two-tailed Student t tests were used to assess statistical significance. *P < 0.05. The underlying data for this figure can be found in (A) The KD of FCHSD2 in control or FCHSD2 siRNA-treated H1975 cells. (B) Endocytic recycling of EGFR was measured in control or FCHSD2 siRNA-treated H1975 cells. Cells were pulsed for 10 min or 30 min with 20 ng/ml biotinylated EGF, stripped, and re-incubated at 37 °C for the indicated times before measuring the remaining intracellular EGF. Percentage of recycled EGF was calculated relative to the initial loading. Data represent mean ± SEM ((TIF)Click here for additional data file.S3 FigCells were incubated with 1 μg/ml HGF for 30 min at 4 °C, washed, and re-incubated at 37 °C for the indicated times. Scale bar, 25 μm. Quantified results are shown in (TIF)Click here for additional data file.S4 FigCells were incubated with 1 μg/ml HGF for 30 min at 4 °C, washed, and re-incubated at 37 °C for the indicated times. Scale bar, 25 μm. Quantified results are shown in (TIF)Click here for additional data file.S5 FigH1975 control or FCHSD2 siRNA-treated cells were incubated with EGFR inhibitor (afatinib) or MET inhibitor (crizotinib) as indicated concentration for 24 h. EGFR, epidermal growth factor receptor; FCHSD2, FCH/F-BAR and Double SH3 Domain-Containing Protein; MET, proto-oncogene c-Met; RTK, receptor tyrosine kinase; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF)Click here for additional data file.S6 FigThe HCC4017 cells stably expressing shCtrl or shFCHSD2 at steady state. ERK1/2, extracellular signal-regulated kinase 1 and 2; FCHSD2, FCH/F-BAR and Double SH3 Domain-Containing Protein; MET, proto-oncogene c-Met; shCtrl, control shRNA; shRNA, small hairpin RNA.(TIF)Click here for additional data file.S7 FigRab7 KD by a different pool of siRNAs abolishes the MET up-regulation induced by FCHSD2 depletion. FCHSD2, FCH/F-BAR and Double SH3 Domain-Containing Protein; KD, knockdown; MET, proto-oncogene c-Met; Rab7, Ras-related protein Rab-7A; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF)Click here for additional data file. 12 Feb 2020Dear Sandy, Thank you for submitting your manuscript entitled "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. 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Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS BiologyCarlyle House, Carlyle RoadCambridge, CB4 3DN+44 1223–442810 11 Mar 2020Dear Sandy,Thank you very much for submitting your manuscript "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.As you will see, all the reviewers are very positive and find the conclusions interesting and significant for the field, however they also raise a few points that would need to be addressed with additional experiments to strengthen the results. 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Please don't hesitate to contact us if you have any questions or comments.Best wishes,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS BiologyCarlyle House, Carlyle RoadCambridge, CB4 3DN+44 1223–442810---------------------------------------------------------------Reviewers’ commentsRev. 1:In the present manuscript, Xiao G. et al., characterized the role of FCHSD2 in endocytosis and trafficking of RTKs. Authors reported that FCHSD2 has a role in ligand-induced trafficking of EGFR and Met receptors, and, in particular, they proposed that it acts as a positive regulator of receptor recycling. They also characterized the role of FCHSD2 in unstimulated basal conditions, where depletion of FCHSD2 causes the increased transcription of EGFR and MET mRNA, possibly through the activation and nuclear translocation of ERK1/2/c-Jun. They also showed that Rab7 is required for the decreased recycling of TfR and EGFR upon siFCHSD2, suggesting that FCHSD2 function could be that of counteracting Rab7. In addition, siRab7 also reverted the increase in EGFR and MET levels induced upon FCHSD2 depletion. FCHSD2 loss correlates with high grade NSCLC; patients with high FCHSD2 have an elevated survival at variance with patients with high Rab7 that display low survival rates.The manuscript provides novel insight into the connection between endocytosis and signaling in cancer. Therefore, is of high interest for the cell biology and cancer community. However, there are a number of issues that need to be solved prior publication.1) The assay described in Fig. 1B, C does not measure properly recycling, but it just detects disappearance of internalized Tf/EGF signal. For TfR, which is almost exclusively recycled back to the PM, it could be used as a proxy for recycling. However, this is not the case for EGFR, which is both recycled and degraded after internalization. Thus, the experiment in Fig. 1C suggests that disappearance of internalized EGF signal is delayed, which is compatible with either defective recycling or defective degradation. This methodological issue need to be better discussed. In addition, the nomenclature on the Y axis in Fig. 1B, C need to be changed accordingly. As it is represented now, it is misleading as it seems that recycling decreases with time.2) Fig. 1F and Fig. 2B. It is not clear how the assay is performed. Are all samples (from 0.5h to 6h of EGF stimulation) treated with cycloheximide for the same period of time? As cycloheximide could really affect the biosynthesis of many factors involved in endocytosis, trafficking and degradation , I would recommend to repeat EGFR degradation curve (-/+ siFCHSD2) without this treatment.3) Fig. 3B. The treatment with Afatinib is not convincing, as it is not substantially reducing EGFR activation (measure as p-EGFR). More consistent inhibition is required to exclude a role for EGFR kinase activity in the process. Also, the WB shown on the left does not reproduce the increase in EGFR and MET upon siFCHSD2 observed in previous experiments. As Erk1/2 is involved in the process, another standard loading control should be used.4) Fig. 3D. Authors concluded from this experiment that STAT3 is not involved in the process. However, its phosphorylation is affected upon siFCHSD2. Authors should better discuss their conclusion.5) Fig. S1. The inhibition of Erk1/2 with SCH compound has the same effect of the Rab7 inhibition on TfR recycling . It seems that the decrease in TfR recycling upon siFCHSD2 requires the action of Erk1/2, as it is reverted by SCH treatment. I did not understand how the authors interpret this results in Fig. S1 at variance with results shown in Fig 5 A.In addition, in Fig. S1, the effects of SCH treatment are mild - if any - as well as the effects of siFCHSD2 in ARPE-19 cells. The magnitude is minimal and, although some time points are statistically significant, it is hard to draw conclusion on the biological significance of these effects. In my opinion, these data on SCH treatment should be more critically discussed.6) Fig. 4F. The WB provided is not completely convincing. The levels are variable and there are some inconsistencies. For instance, MET levels seem not to be rescued by both treatments in H1975, just all levels are decreased. Similarly, in HCC4079, MET levels are variable, sometimes increasing (SCH) sometimes decreasing (GSK) after treatments. It is not clear what these treatments are really doing. In addition, the blots are not representative of the quantification on the right. In the quantitation, samples should be all normalized to the same reference, i.e. the not treated Ctrl sample.An important experiment that would corroborate this finding is the analysis of EGFR and MET mRNA levels upon siFCHSD2, to investigate if they are rescued by these treatments.7) The effect of siRab7 are strong and convincing. However, it is not clear how Rab7 function is linked to Erk1/2 nuclear translocation and activation, and to the induced EGFR/MET transcription. The model shown in Fig. 6A is too complicated and does not convey the message.Which is the mechanism though which Rab7 KD revert the increased EGFR and MET levels? Authors should show if siRab7 is able to revert the MET and EGFR mRNA levels.Does siRab7 revert the increased Erk1/2, c-Jun, Ets-1 levels in the nucleus?These connections need to be investigated and further discussed in the text.Rev. 2:This is an excellent study interrogating how membrane trafficking aletrations downstream of FCHSD2 depletion impact ERK1/2 signaling. The primary conclusions are that FCHSD2, by negative regulation of Rab7, diverts tyrosine kinase receptors (TKRs) from late endosomes, limiting access of TKRs to ERK1/2 activation at the late endosome. Loss of FCHSD2 increases traffic of TKRs to LE and subsequent activation of ERK1/2. The data, for the most part, support the stated conclusions. My main comments are as follows.1. The authors should be more attentive to alerting the reader what was done in the KRAS/p53 line and what was done in EGFR mutant. For the non-cancer specialist, the potential importance of the distinction might be lost. The authors might also consider adding a discussion paragraph on the topic of the oncogenic driver mutation and the impact of FCHSD2 (loss thereof).2. The authors might consider extending the main conclusion, that KD of FCHSD2 increases expression of EGFR and MET, in analyses of other human NSCLC cell lines, both EGFR mutant and KRAS.3. The authors might provide some speculation (or perhaps data) on how FCHDS2 acts as a negative regulator of Rab7. Does FCHSD2 activate a Ran7 GAP or inhibit a Rab7 GEF?4. Fig 6 is not particularly strong, and I do not feel it strengthens the paper but rather detracts from the otherwise strong cell biology. To appreciate the data in panel B one would want to see expression of FCHSD2 in adjacent normal lung tissue. Pushing a link between the expression of a single protein (FCHSD2) and grade is not particularly compelling. I assume the analyses in Fig6C are based on RNAseq. Have they correlated IHC with RNAseq in their own cohort (panel B) to establish the RNAseq data agrees with the IHC? The author's need to present data on the expression distribution of FCHSD2 and Rab7 so the reader can assess how meaningful the distinction is. Did the author's use above and below the median or upper and lower quartiles? They should also look at the TCGA database to determine how specific this is for lung.5. Throughout the manuscript the authors should reanalyze the recycling curves. In many instances it looks like the extent of recycling rather than the rate of recycling is changed. For example, in Fig 1B it is the extent of transferrin receptor recycling that is changed by FCHSD2 depletion not the rate, suggesting a fraction of transferrin receptor is trapped and no longer recycled rather than a slowing of recycling. Although I do not think it will change the conclusions it might change how one thinks about the molecular mechanism(s) underlying the effects.Rev. 3: Peter S McPherson – Please note that this reviewer has waived anonymityThis is a clear, concise, well-executed study, exactly what we have come to expect from Dr. Schmid and her colleagues. I was left a little wanting, how exactly does the FCHSD2-depeletion induced upregulation of the RTKs occur, what specifically does Rab7 do in this pathway, why the seeming conundrum between increased trafficking to late endosome/lysosome and yet upregulation of expression? But there is valuable information in this study and no one study ever has all the answers.Abstract. "…can reciprocally regulate endocytic trafficking by creating feedback loops in non-small-cell lung cancer (NSCLC) cells…". The sentence is awkward. Presumably these feedback loops are present in other cancer cells, perhaps in non-cancer cells.Line 53, can you please be more specific as to what you mean by "…cancer-specific activation…", in particular, what is meant by activation?Figure 1 and throughout, use 2 siRNAs or better describe the specificity of the single one used or perhaps validate 1-2 key phenotypes in a knockout line.Figure 1D, please, a word or two about the specificity of the PO4-EGFR antibody in the results section.The upregulation of mRNA in Figure 3 is relatively minor, and in light of the enhanced degradation its seems the balance should not be towards such a strong upregulation of the protein. Please discuss.Figure 5, one Rab7 siRNA?Figure 5, higher levels of active Rab7 in FCHSD2 knockdown cells. It would be nice if there was some additional data here. What explains the activation, regulation of a GEF or a GAP?Figure 5. There is some uncertainty regarding the specificity of the antibodies recognizing the active Rabs. The results state that an antibody specific to active Rab7 was used without reference or validation. The authors need to address this issue. 1 May 2020AttachmentResponse to reviewers-final.pdfSubmitted filename: Click here for additional data file. 22 May 2020Dear Sandy,Thank you for submitting your revised Research Article entitled "FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking" for publication in PLOS Biology. I have now obtained advice from the original Academic Editor, who has assessed the revision along with the team of editors.We're delighted to let you know that we're now editorially satisfied with your manuscript. However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. 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In response to tumor signals, mesenchymal stem cells (MSCs) are recruited to tumor sites and activated to promote tumor progression. Emerging evidences suggest that in addition to tumor cells, non-tumor cells in tumor microenvironment could also interact with MSCs to regulate their phenotype and function. However, the mechanism for MSCs regulation in gastric cancer has not been fully understood. In this study, we reported that tumor-educated neutrophils (TENs) induced the transformation of MSCs into cancer-associated fibroblasts (CAFs) which in turn remarkably facilitated gastric cancer growth and metastasis. Mechanistic study showed that TENs exerted their effects by secreting inflammatory factors including IL-17, IL-23 and TNF-α, which triggered the activation of AKT and p38 pathways in MSCs. Pre-treatment with neutralizing antibodies to these inflammatory factors or pathway inhibitors reversed TENs-induced transformation of MSCs to CAFs. Taken together, these data suggest that TENs promote gastric cancer progression through the regulation of MSCs/CAFs transformation. Gastric cancer (GC) is one of the most common malignant tumors worldwide. Although the recent advances in curative resection and targeted therapy, the overall survival rate of patients with GC is still poor with a 5-year survival rate of 20–40% . The mecNeutrophils are the first defense against infection and tissue damage. The studies over the past decade have revealed an important role of neutrophils in the pathogenesis of many cancers . NeutropNon-tumor cells within tumor microenvironment (TME) interact with each other and form an intricate network that is involved in tumor development and progression. The previous studies have shown that cancer-associated fibroblasts (CAFs) facilitate cancer growth and metastasis in various cancers including GC . Furtherin vitro and accelerated GC growth and metastasis in vivo. The induction of MSCs/CAFs transformation represents a new mechanism for the pro-tumor roles of neutrophils in cancer.In this study, we reported that tumor-educated neutrophils (TENs) could mediate the transformation of MSCs to CAFs by secreting inflammatory factors. MSCs activated by TENs promoted the proliferation, migration and invasion of GC cells Mesenchymal stem cells were isolated from human umbilical cord and characterized as previously described . Cells a6) were seeded in RPMI 1640 medium  containing 10% (v/v) FBS and 1% penicillin/streptomycin.Peripheral blood samples were collected from healthy volunteers and the study was approved by the ethics committee of Jiangsu University (2014280). Neutrophils were isolated by using Polymorphprep  as previously described . RBCs weFirstly, GC cells were cultured in DMEM with 10% FBS. When reached 80%, cells were changed to new serum-free medium. After 48 h of incubation, the conditioned medium of gastric cancer cells (GC-CM) was collected and centrifuged to remove cell debris. The CM was stored in −80°C refrigerator until use. Secondly, neutrophils were treated with GC-CM (1:1 ratio) for 24 h. Then, neutrophils were collected, washed with PBS (to remove factors present in GC-CM), and re-seeded in new serum-free medium for 48 h. The CM of tumor-educated neutrophils (TEN-CM) was collected, centrifuged to remove cell debris and stored in −80°C refrigerator until use. Finally, MSCs were co-cultured with TEN-CM (1:1 ratio) for 24 h. Then, MSCs were collected, washed with PBS (to remove factors present in TEN-CM), and re-seeded in new serum-free medium for 48 h. The CM of TEN-CM-primed MSCs was collected, centrifuged to remove cell debris and stored in −80°C refrigerator until use.Human GC cells were plated in 6-well plates at 800 cells per well and allowed to attach overnight. The cells were then treated with CM from TENs-activated MSCs for 24 h. Then, the CM was changed and added new culture medium for the next 7 days. Finally, the cells were fixed in 4% paraformaldehyde, stained with crystal violet, and photographed under a microscope. The experiments were performed in triplicate for each group.2. Then, different CMs were added into the culture plate and cells were treated for different times. The culture medium was discarded and CCK8 was added into each well for the last 4 h. The absorbances of each well were read at 450 nm using an enzyme-linked immunosorbent plate assay reader . The experiments were performed in triplicate for each group.The proliferative abilities of GC cells and MSCs were determined by CCK8  assay according to the manufacture’s instruction. Cells were seeded in 96-well plates at 3,000 cells per well and allowed to attach overnight at 37°C in 5% CO4) treated with different CMs for 24 h were seeded in the upper chamber in serum-free medium. Then, 600 μL of complete medium was added to the lower chamber and the cells were incubated for 24–48 h at 37°C in 5% CO2. Cells which remained on the top side of the membrane were wiped off with cotton swabs. The migrated or invaded cells on the bottom side of the membrane were fixed in 4% paraformaldehyde and stained with crystal violet. The number of migrated or invaded cells was counted under a microscope. At least six fields from each group were selected.Cell migration and invasion assays were tested in transwell chemotaxis chambers . Briefly, an appropriate 50 microliters of Matrigel  was added into the upper chamber of the transwell plates for the invasion assay, while the plates without Matrigel in the upper chamber were used for the migration assay. Cells  according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed into cDNA by Reverse Transcription System . To quantify the mRNA levels of inflammatory factors, real-time PCR was performed using SYBRGreen Kit  on a Bio-Rad CFX96 Detection System. The relative gene expression was normalized to β-actin. Data were analyzed by using the comparative Ct method. The primers of target genes used in this study were listed in The total protein from GC cells and MSCs were obtained with RIPA lysis buffer containing protease inhibitor cocktail . Proteins from each group were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies including FAP , α-SMA , β-actin , AKT/p-AKT , p38/p-p38 , STAT3/p-STAT3 , p65/p-p65 , ERK/p-ERK , N-cadherin , E-cadherin , Cyclin D1 , and PCNA . The membranes were washed with TBST and incubated with secondary antibodies. The secondary antibodies were HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies . The bands were visualized by ECL reagent and the β-actin was served as the loading control.6 cells in 100 μL PBS per mouse) pre-treated with different CMs for 24 h were subcutaneously injected into the flank of mice (n = 5/group). The volume (V) and weight of tumor were assessed every 5 days, and tumor volumes were calculated using the formula: V = 0.5 × a × b2, where V represents volume, a represents longitudinal diameter and b represents latitudinal diameter. To examine the metastatic ability of GC cells in vivo, the same number of GC cells was injected intraperitoneally into mice. All mice were killed at 4 weeks after injection, the metastases of tumor to different organs were observed and the number of visible metastatic nodules was recorded. The protocol was approved by the Animal Use and Care Committee of Jiangsu University (2014280).All animal studies were performed by using 4–6 week female BALB/c nude mice . GC cells  followed by incubation with the secondary antibody for 30 min at room temperature and stained with H&E, respectively. The histological changes of tumor tissue were examined under an optical microscope .Tumor tissues were fixed with formaldehyde, embedded in paraffin and then cut into 5 μm sections. Briefly, the slides were dewaxed in xylene and rehydrated in graded ethanol solutions. Then, the sections were soaked in HImmunofluorescence was performed to determine the expression of proteins related to CAFs markers. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, then blocked with 5% bovine serum albumin, and incubated with goat anti-rabbit FAP  and goat anti-mouse α-SMA antibody  at 4°C overnight. The MSCs were then washed and incubated with Alexa Fluor 555 conjugated donkey anti-rabbit IgG or FITC conjugated goat anti-Rabbit IgG  for 1 h. The nuclei were stained with DAPI  and images were acquired using a fluorescent microscope . The experiments were repeated in triplicate for each group.t test for two groups were applied for statistical analysis. P < 0.05 was considered statistically significant.Statistical analyses were carried out by GraphPad Prism Software (version 4). All values were presented as mean values ± SD. Two-way ANOVA for multiple groups and unpaired Student’s in vitro.We first wanted to know whether TENs could activate MSCs. Human peripheral blood derived neutrophils were treated with the supernatant from GC cells and the CM of primed neutrophils was collected. After then, MSCs isolated from human umbilical cord were treated with TEN-CM for 24 h and the alterations in their phenotype and function were examined. The results of western blot showed that treatment with CM from TENs induced the expression of CAFs markers, including fibroblast activating protein (FAP) and α-smooth muscle actin (α-SMA), in MSCs . QuantitTo clarify the mechanism for MSCs activation by TENs, we treated MSCs with different TEN-CMs for 24 h and measured the responses of cell signaling pathway. TEN-CM treatment increased the expression of phosphorylated AKT (p-AKT) and p38 (p-p38) in MSCs and the statistical analysis of western blot , indicatWe then wanted to know the functional roles of MSCs/CAFs transformation induced by TENs. MSCs were treated with TEN-CM and the culture supernatant from MSCs was collected to act on GC cells for 24 h. We first tested the expression of proliferation and migration related proteins and genes in GC cells. We found that the expression of N-cadherin, Cyclin D1 and PCNA was increased, and the expression of E-cadherin was decreased in GC cells after treatment . Then, tTo explore which molecules released by TENs induce MSCs/CAFs transformation, we measured the expression of inflammatory factors in neutrophils after treatment with the supernatant from GC cells. Several genes of interest such as IL-17, IL-23, and TNF-α were identified to be highly expressed in the treated neutrophils . We chosin vivo, we established mouse peritoneal metastasis model. We found that treatment with culture supernatant from TEN-CM-primed MSCs promoted the colonization of GC cells in the intestines .JZ, CJ, and HS completed all the experiments and wrote the manuscript. WX, HQ, and XZ designed the outline of the experiments. WL, ZM, YS, and RJ reviewed and corrected the draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.